JP2007215513A - Gene associated with stress resistance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene taking part in an active oxygen-removing system in a cell; and to provide a recombinant plant having active oxygen stress-resistance imparted by introducing the new gene into the plant. <P>SOLUTION: A DNA encoding a protein newly isolated from tobacco and having a specific amino acid sequence, a DNA having a specified base sequence or analogues of these DNAs are used as the gene taking part in the active oxygen-removing system in the cell. The genes are introduced into the chromosomal DNA of the plant. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to genes involved in stress tolerance, particularly genes that are involved in active oxygen stress tolerance and can confer resistance to various environmental stresses such as peroxides, heavy metal salts, high salt concentrations, and drying. .

  When living organisms such as plants are subjected to environmental stresses such as heavy metals, high salt concentrations, and drying, the production of active oxygen that damages macromolecular substances such as proteins, lipids, and nucleic acids increases in the cells ( Non-patent document 1). Various intracellular reactions caused by the increase of active oxygen are called active oxygen stress, and the cells of organisms have countered this by providing an active oxygen removal system. Various enzymes that work in the oxygen scavenging system and genes encoding these enzymes have been reported.

Among them, the YAP1 gene isolated as a transcription factor having a DNA binding activity similar to that of AP-1 transcription factor from yeast ( Saccharomyces cerevisiae , hereinafter simply referred to as yeast) is a central component in the reactive oxygen removal system. More than 20 genes considered to be related to the active oxygen removal system, such as thioredoxin gene (TRX2) and γ-glutamylcysteine synthetase gene (GSH1), are known to be YAP1 gene. (Non-Patent Document 2).

  Therefore, yeast that has disrupted the YAP1 gene can be used as an active oxygen stress inducer, such as peroxides such as hydrogen peroxide, reduced glutathionates such as diamide, and / or electrophilic substances such as diethylmaleic acid. On the other hand, in normal yeast, an increase in the expression level of this gene is observed in the presence of these substances. In addition, a cDNA library of Arabidopsis thaliana was inserted into an expression vector for yeast, introduced into yeast cells in which the YAP1 gene was disrupted, and screened using thiol oxidant diamide resistance as an index. From the resulting resistant strain, NADPH oxidoreductase Has also been reported (Non-patent Document 3).

  Furthermore, the YAP2 gene is also isolated as a homologous gene of the YAP1 gene, and it is clear that a strain in which both the YAP1 gene and the YAP2 gene are disrupted (double disruption strain) is highly sensitive to hydrogen peroxide and heavy metals. (Non-Patent Document 4).

With the accumulation of such knowledge, in recent years, it has been attempted to introduce a gene involved in the above-mentioned active oxygen removal system into a plant to create a plant having resistance to active oxygen stress. For example, an active oxygen removal system in higher plants. It has also been reported that hybrid aspen introduced with a gene for guaiacol peroxidase (GPX), which is an enzyme that acts on the plant, has acquired resistance to active oxygen stress, thereby obtaining individuals exhibiting resistance to the herbicide paraquat ( Non-patent document 5)
Morita et al., "Protein Nucleic Acid Enzyme", 1999, vol.44, p.2322-2238 Lee et al., “Journal of Biological Chemistry” (USA), 1999, vol. 274, p. 16040-16046. Babiychuk et al., “Journal of Biological Chemistry”, (USA), 1995, vol.270, p.26224-26231 Hirata et al., “Molecular and General Genetics”, (Germany), 1994, vol. 242, pages 250-256. Kawaoka et al., “Plant Physiol.” (USA), 2003, vol. 132, p. 1177-1185

  The present invention isolates a novel gene involved in the intracellular reactive oxygen scavenging system, and newly introduces a recombinant plant imparted with resistance to active oxygen stress by introducing the isolated gene into a plant. It was done for the purpose of creating.

In order to solve the above-mentioned problems, the present inventors have conducted intensive research and found novel genes CDR1 and CDR2 that restore the resistance to peroxide and heavy metal of a double gene disrupted strain in which both YAP1 gene and YAP2 gene are disrupted. The present invention was completed by succeeding in isolation from tobacco ( Nicotiana tabacum ).

  That is, the present invention provides DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or at least one amino acid is deleted, substituted, and / or added to these amino acid sequences. The present invention relates to a DNA that encodes a protein having an amino acid sequence and imparts hydrogen peroxide resistance to the host cell when introduced into the chromosomal DNA of the host cell. The present invention also relates to DNA having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or when hybridizing with these base sequences under stringent conditions and introduced into the chromosomal DNA of the host cell. And a DNA that imparts hydrogen peroxide resistance to the host cell. Furthermore, the present invention relates to a gene introduction vector having the above DNA, a transformed cell in which the DNA is introduced into chromosomal DNA, a transformed plant individual obtained by the growth and differentiation of the transformed cell, and the DNA Relates to the protein encoded by.

  According to the present invention, novel genes CDR (Nicotiana tabacum cadmium-resistant) 1 and CDR2 involved in the intracellular reactive oxygen elimination system are provided. By introducing this CDR1 gene or CDR2 gene into the chromosomal DNA of the cell, a transformed cell having resistance to active oxygen stress and exhibiting resistance to various environmental stresses such as peroxide, heavy metal salt, high salt concentration, and drying, and / Or transformed individuals can be obtained. In addition, by further analyzing the structure and function of the CDR1 gene and / or useful information about the mechanism of the active oxygen removal system in the cell can be obtained.

  The present invention also provides a vector having the CDR1 gene or CDR2 gene. By introducing this vector into the cell, a novel gene involved in the active oxygen removal system is introduced into the chromosomal DNA of the cell, and has resistance to active oxygen stress, peroxide, heavy metal salt, high salt concentration, drying, etc. Transformed cells showing resistance to various environmental stresses and / or transformed individuals can be obtained.

  According to the present invention, there is also provided a transformed cell in which the CDR1 gene or CDR2 gene is introduced into its chromosomal DNA. By growing and differentiating the transformed cells, a transformed individual having resistance to active oxygen stress and resistance to various environmental stresses such as peroxide, heavy metal salt, high salt concentration, and drying can be obtained.

  Moreover, according to this invention, the transformed plant individual obtained by the said transformed cell growing and differentiating is provided. Therefore, a novel gene involved in the active oxygen removal system has been introduced into the chromosomal DNA of the cell, and it has resistance to active oxygen stress, and exhibits resistance to various environmental stresses such as peroxide, heavy metal salt, high salt concentration, and drying. Convertible plant individuals can be obtained. Such transformed plants grow in environments that have traditionally been difficult to grow, such as salt-enriched soils and dry land, preventing the expansion of wasteland and deserts, greening and afforestation of these land, and even the earth. It can play a big role in preventing global warming.

  In addition, according to the present invention, a protein encoded by the CDR1 gene or CDR2 gene is provided. By allowing this protein to function in the cell, the active oxygen stress resistance is imparted to the cell and the individual produced by the proliferation and differentiation of the cell, and as a result, peroxide, heavy metal salt, high salt concentration, dryness, etc. Resistance to various environmental stresses can be imparted. In addition, by further analyzing the structure and function of this protein, useful information can be obtained about the mechanism of the active oxygen removal system in the cell.

  The present invention is described in detail below.

  The DNA according to the present invention encodes a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. The novel gene CDR1 involved in the active oxygen scavenging system is composed of DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2, while the gene CDR2 encodes a protein having the amino acid sequence shown in SEQ ID NO: 4. It consists of DNA. Such DNA may be isolated as DNA from cells such as plants, or may be obtained by reverse transcription of cDNA from RNA after isolation as RNA. When isolated as RNA, cells previously exposed to active oxygen stress such as hydrogen peroxide may be used as the material. However, the DNA can also be obtained by chemical synthesis. Methods known to those skilled in the art can be used as methods for isolating DNA and RNA from cells and methods for chemically synthesizing DNA.

  For example, in order to obtain a DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 as RNA from tobacco cells and then reverse transcribe cDNA from the RNA, first, whole tobacco, Or, a tissue such as tobacco leaves, stems, roots, flowers and callus or a part thereof is frozen in liquid nitrogen, and then ground in a mortar, and then from this ground product, acid guanidine phenol chloroform method, guanidine thiol The crude RNA fraction was extracted using the cinnanate-cesium chloride method, glyoxal method, lithium chloride-urea method, proteinase K-deoxyribonuclease method, etc., and the obtained crude RNA fraction was extracted with oligo dT-cellulose or Sepharose 2B. Purified by affinity column method or batch method using poly-U-Sepharose etc. Obtaining a (poly A + mRNA). This mRNA may be further fractionated by sucrose density gradient centrifugation or the like.

  Next, cDNA is reverse-transcribed from the purified mRNA obtained as described above using, for example, the Hoffman method. Specifically, after using a commercially available kit (for example, ZAP-cDNA Synthesis Kit or HybriZAP 2.1 XR Vector Cloning Kit (both manufactured by Stratagene)), oligo dT is added to the purified mRNA as a primer. After the reverse transcriptase is allowed to act to form a DNA-RNA hybrid, the RNA strand is degraded with RNase H, and then the DNA polymerase I is allowed to act so that the double-stranded cDNA in which the purified mRNA is reverse transcribed is obtained. Obtainable.

  DNA encoding the protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is prepared by appropriately adding an adapter such as EcoRI-NotI-BamHI adapter to both ends of the double-stranded cDNA, and then, for example, a HybriZAP 2.1 XR vector ( Stratagene, etc.) and linked to a cloning vector and packaged into phage particles. A cDNA library is prepared, and the complementation test using the yeast in which the YAP gene has been disrupted as a host is performed on the libraryed cDNA. It can be obtained by screening.

  That is, the transcriptional activity of a plasmid, such as the pAD-GAL4 plasmid, which is capable of autonomous replication of the libraryed cDNA in yeast and has an effective transcription activation domain (for example, GAL4 activation domain). After linking to the downstream of the activation domain, this is introduced into the yeast in which the YAP1 gene and / or YAP2 gene has been disrupted using the lithium acetate method, protoplast method, electroporation method, electroinjection method, etc. Are cultured in a medium containing 4 mM hydrogen peroxide or 150 μM cadmium ions. YAP1 gene and / or YAP2 gene disrupted yeast is inhibited from growing in a medium containing hydrogen peroxide or cadmium ions at the above concentrations. Thus, it is considered that the function of the disrupted YAP1 gene and / or YAP2 gene is complemented, and the active oxygen removal system to counter active oxygen stress caused by the presence of hydrogen peroxide and cadmium ions is restored. Therefore, if such a strain is selected and the introduced cDNA is recovered, the gene CDR1 or CDR2 involved in the intracellular reactive oxygen elimination system, that is, a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 Can be obtained.

  In addition, it is preferable to use the strain which destroyed both YAP1 gene and YAP2 gene as yeast used for the said screening. In a strain in which only one of the YAP1 gene or the YAP2 gene is disrupted, sufficient growth can be achieved between normal strains that retain both of these genes even when cultured in a medium supplemented with hydrogen peroxide or cadmium. Is difficult to detect (Hirata et al., Mol. Gen. Genet., Vol. 242, p. 250-256, 1994). The yeast in which the YAP1 gene and / or the YAP2 gene is disrupted is preliminarily inserted into the normal YAP1 structural gene and / or YAP2 structural gene into which the HIS3 gene, URA3 gene, LEU2 gene, TRP1 gene or the like has been inserted and whose function has been disrupted It can be obtained by replacing the YAP1 structural gene and / or YAP2 structural gene in the chromosomal DNA of yeast using homologous recombination or the like.

  Confirmation of the base sequence of the cDNA recovered as a result of the above screening is performed by cleaving this cDNA with a restriction enzyme, religating it to an appropriate plasmid, subcloning, and then subjecting it to a Maxam-Gilbert chemical modification method or a dideoxynucleotide chain termination method ( F. Sanger et al., Proc. Natl. Acad. Sci. USA, vol. 74, P. 5463-5469, 1977) etc., and a commercially available automatic base sequencer (for example, CEQ2000 DNA sequencer manufactured by BECKMAN) It can be performed appropriately.

  The base sequence of DNA shown in SEQ ID NO: 1 or SEQ ID NO: 3 is determined as described above. This DNA encodes the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and is considered to be involved in the intracellular active oxygen removal system and to impart resistance to active oxygen stress due to peroxides, heavy metals, and the like.

  However, it is normal that the base sequences of organism genes that have the same function are somewhat different from each other and from individual to individual. Therefore, even if a protein having the same amino acid sequence as the above amino acid sequence is not given, or even if it is not identical to the base sequence of the above DNA, the DNA according to the present invention is resistant to hydrogen peroxide to the host cell into which it has been introduced. As long as it is given, it may encode a protein having an amino acid sequence in which at least one amino acid is deleted, substituted, and / or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Alternatively, it may be one that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions. In this case, the requirement for imparting hydrogen peroxide resistance is that the gene involved in the active oxygen removal system is an inducer of active oxygen stress, as is clear from the YAP1 and YAP2 genes of yeast. This is because it is considered to impart resistance to peroxide such as hydrogen peroxide which is a substance.

  The DNA of the present invention encoding a protein having an amino acid sequence in which at least one amino acid is deleted from the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 is shown in SEQ ID NO: 2 or SEQ ID NO: 4. A DNA encoding an amino acid sequence from which about 1 to 20 amino acids have been deleted is preferable, and a DNA encoding an amino acid sequence from which about 1 to 5 amino acids have been deleted is particularly preferable. .

  In addition, the DNA of the present invention encoding a protein having an amino acid sequence in which at least one amino acid is substituted with respect to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 includes SEQ ID NO: 2 or SEQ ID NO: 4. DNA encoding an amino acid sequence in which about 1 to 160 amino acids are substituted with other amino acids relative to the amino acid sequence shown is preferred, and in particular, amino acids in which about 1 to 40 amino acids are substituted with other amino acids. DNA encoding the sequence is preferred.

  Furthermore, the DNA of the present invention encoding a protein having an amino acid sequence to which at least one amino acid is added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 includes SEQ ID NO: 2 or SEQ ID NO: 4. A DNA encoding an amino acid sequence to which about 1 to 20 amino acids are added to the amino acid sequence shown is preferable, and a DNA encoding an amino acid sequence to which about 1 to 5 amino acids are added is particularly preferable.

  On the other hand, the DNA of the present invention that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions has, for example, a formamide concentration of 30 to 50 W / V%, preferably 50 W / V%, A DNA that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under a temperature of 37-50 ° C, preferably 42 ° C is preferred.

  DNA encoding a protein having an amino acid sequence in which at least one amino acid is deleted, substituted and / or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO: 1 Alternatively, the DNA that hybridizes with the base sequence shown in SEQ ID NO: 3 under stringent conditions is, for example, DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO: 1 or SEQ ID NO: DNA or cDNA isolated from tobacco cells or other plant cells, microbial cells, or animal cells, using the DNA having the base sequence shown in 3 as a probe, and the above hybridization conditions, ie, formamide concentration of 30 to 50 % W / V, preferably 50 W / V%, temperature 37-50 ° C., preferably 42 By performing the hybridization in, it can be obtained.

  Further, a DNA encoding a protein having an amino acid sequence in which at least one amino acid is deleted, substituted, and / or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence DNA that hybridizes under stringent conditions with the nucleotide sequence shown in No. 1 or SEQ ID No. 3 is, for example, DNA encoding a protein having the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4, or SEQ ID No. 1 or It can also be prepared by artificially introducing a site-specific mutation into a DNA having the base sequence shown in SEQ ID NO: 3 using a known method such as the Kunkel method or the Gapped duplex method or a method analogous thereto. Mutation introduction kits for simply introducing such site-specific mutations are also commercially available (for example, TAKARA, Mutant-K, Mutant-G, or LA PCR in vitro Mutagenesis series kits, etc. ).

  The gene introduction vector of the present invention comprises the DNA of the present invention, that is, the DNA encoding the protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. On the other hand, DNA encoding a protein having an amino acid sequence in which at least one amino acid is deleted, substituted, and / or added, DNA having a base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO: It has a DNA that hybridizes with the base sequence shown in 1 or SEQ ID NO: 3 under stringent conditions.

  Such a vector is obtained by cleaving the DNA with an appropriate restriction enzyme, and then converting the DNA into various known DNA elements required for achieving the object of the present invention, such as cis elements such as promoters and enhancers. Ribosome binding sequence (SD sequence), splicing signal, poly A addition signal, terminator, selection marker gene, etc., and restriction enzyme sites of known vectors that can be replicated in the host cell into which these are to be introduced, or multicloning sites It can produce by connecting.

  The promoter may be any promoter as long as it can express the DNA. When Escherichia coli is used as a host, trp promoter derived from Escherichia coli or phage, lac promoter, In addition to the PL promoter, the PR promoter, and the like, it is preferable to use a promoter such as the tac promoter obtained by artificially modifying these promoters. When yeast is used as a host, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like are preferable. When plant cells are used as hosts, the 35S promoter derived from cauliflower mosaic virus (CaMV), the MC8 promoter derived from Astragalus dwarf virus, the rbcS promoter, the ubiquitin promoter, the promoter of nopaline synthase (NOS) gene, octopine ( An OCT) synthase gene promoter is preferred. Furthermore, when using an animal cell as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like are preferable. In animal cells, the human cytomegalovirus early gene promoter and the like can also be used.

  Examples of known vectors for ligating the DNA of the present invention and DNA elements such as the above promoter include phage DNA such as λ phage, plasmid DNA for E. coli such as pBR322, pBR325, pUC118, and pUC119, pUB110, Plasmid DNA for Bacillus subtilis such as pTP5, plasmid DNA for yeast such as YEp13, YEp24, and YCp50, plasmid DNA for binary vectors such as pBI101, pBI121, pBI2113, pBI2113Not, pBI221, pBIG, pGA482, pGAH, or pLGV23Neo And plasmid vectors for plant cells of intermediate vectors such as pMON200 and pNCAT. In addition, insect virus vectors such as baculovirus, and animal virus vectors such as retrovirus and vaccinia virus can also be used as vectors for ligating the DNA. When a gene is introduced into a plant cell using a binary vector-based plasmid DNA for plant cells as a vector, it is necessary to link the DNA between the boundary sequences LB and RB, that is, the T-DNA region.

The transformed cell of the present invention can be obtained by introducing the DNA of the present invention into the host cell chromosome. In this case, typical host cells include, for example, Escherichia coli Hms174 (DE3) strain, K12 strain or DH1 strain, Bacillus subtilis MI 114 strain, 207-21 strain, Pseudomonas putida ( Pseudomonas putida ), Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens ) C58 strain, LBA4404 strain, EHA101 strain, EHA105 strain or C58C1RifR strain, Agrobacterium lysogenes ( Agrobacterium rhizogenes ) or bacteria such as Rhizobium meliloti , yeasts such as yeast ( Saccharomyces cerevisiae ), Shizosaccharomyces pombe or Pichia pastoris ( Pichia pastoris ), Arabidopsis, Arabidopsis, , Carrot, petunia, poplar, eucalyptus, acacia, cedar Are plant cells such as pine, insect cells such as Sf9 cells or Sf21 cells, or animals such as monkey cells COS-7 or Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells or human FL cells Mention may be made of cells. However, in the present invention, the host cell is not particularly limited. Whatever can express the DNA of the present invention, the transformed cell of the present invention can be obtained by introducing this DNA into the chromosome.

  In order to introduce the DNA of the present invention into the chromosome of the host cell, as described above, the DNA of the present invention is ligated to a DNA element such as a promoter selected according to the host cell and a vector. A vector for gene transfer may be prepared and introduced into each host cell using a known gene transfer method. Examples of gene transfer methods include calcium ion method (Cohen et al., Proc. Natl. Acad. Sci., USA, 69: 2110-2114, 1972), electroporation when bacteria are used as host cells. Method (Becker et al., Methods. Enzymol., 194: 182-187, 1990), freeze-thaw method, tripartite conjugation method (M. Bevan Nucleic Acids Research, 12: 8711, 1984), etc. Electroporation method, electroinjection method, protoplast method, spheroplast method (Hinnen et al., Proc. Natl. Acad. Sci., USA, 75: 1929-1933, 1978), lithium acetate method (Itoh, J. Bacteriol., 153: 163-168, 1983), etc., when plant cells are used as host cells, the Agrobacterium method, electroporation method, polyethylene glycol method (Abel et al., Plant J., 5: 421-427, 1994), particle gun method Liposome method, microinjection method, or the like, animal cells and insect cells when the host cells, electroporation method, calcium phosphate method, the lipofection method can be used.

  The transformed plant individual of the present invention can be obtained by proliferating and differentiating transformed cells obtained by introducing the DNA of the present invention into the chromosomes of plant cells.

  When introducing the DNA of the present invention into the chromosome of a plant cell, as described above, the DNA of the present invention is linked to a DNA element such as a promoter selected for the plant cell and a vector, and then the gene introduction vector of the present invention. May be prepared and introduced into each host cell using a known gene transfer method.

  Regarding the DNA element, as already exemplified, the 35S RNA promoter, MC8 promoter, rbcS promoter, ubiquitin promoter, nopaline synthase (NOS) gene promoter, octopine (OCT) synthase gene promoter are used as plant cell promoters. Etc. can be used. In addition, as a terminator, a terminator derived from cauliflower mosaic virus or a terminator derived from a nopaline synthase gene is used, and as a selection marker gene, resistance to a kanamycin resistance gene (NPTII gene), a hygromycin resistance gene (htp gene), bialaphos. One or more genes selected from a phosphinothricin acetyltransferase (bar) gene that imparts sex can be used. Furthermore, in addition to these DNA elements, for example, chloramphenicol acetyltransferase (CAT) gene, β-glucuronidase (GUS) gene, green fluorescent protein (GFP) gene, etc. may be used as a reporter gene.

  Regarding vectors, host cells, and gene transfer methods, as already exemplified, vectors include pBI101, pBI121, pBI2113, pBI2113Not, pBI221, pBIG, pGA482, pGAH, pLGV23Neo, pMON200, pNCAT, etc. , Arabidopsis thaliana, tobacco, corn, rice, carrot, petunia, poplar, eucalyptus, acacia, cedar or pine etc. As gene transfer methods, Agrobacterium method, electroporation method, polyethylene glycol method, particle gun method, liposome Method, microinjection method and the like can be used. The host cell is a cell of any organ (eg, leaf, petal, stem, root, rhizome, seed, etc.) or tissue (eg, epidermis, phloem, soft tissue, xylem, vascular bundle, etc.). May be used.

  However, in addition to the above DNA elements, vectors, host cells, and gene transfer methods, the DNA of the present invention can be introduced into a chromosome and expressed to produce a transformed cell. Further, this transformed cell can be proliferated and differentiated. Any one can be used in the present invention as long as it can produce a transformed plant individual. Moreover, the selection marker gene may be incorporated into a single vector together with the DNA of the present invention, or may be separately incorporated into two or more vectors.

  The host cell after gene introduction is cultured in a known medium and environmental conditions suitable for the growth and differentiation of the plant cell. In this process, if the transformed cell is selected by using the expression of the selection marker gene introduced together with the DNA of the present invention as an index, and then the plant is proliferated and differentiated to redifferentiate the plant individual, the transformed plant individual of the present invention Can be obtained. The introduction of the DNA of the present invention into the chromosome of a transformed plant individual or a progeny individual thus obtained is performed by extracting the DNA from the cells of the individual according to a conventional method, and using a known method such as a PCR method or Southern hybridization method. This can be confirmed by performing an analysis using this method.

  On the other hand, the expression of the DNA of the present invention in the transformed plant individual of the present invention or a progeny individual thereof can be confirmed and evaluated by known techniques such as RT-PCR method, Northern hybridization method, Western blotting method and the like. . That is, for example, after giving a dry and / or low temperature stress to the individual, a transformed plant individual or its progeny grown under the same conditions, and / or a non-transformed plant individual that did not give such stress. In addition, if the RNA (when using RT-PCR method or Northern hybridization method) or the gene product (when using Western blotting method) is analyzed by these methods, the expression level of the DNA of the present invention can be known. it can. In addition, if it is a plant individual grown in a culture vessel, the drought stress is obtained by extracting the plant from an agar medium and drying it on a filter paper for 10 minutes to 24 hours. Can be provided by placing it in an environment of −4 to 15 ° C. for 10 minutes to 24 hours. On the other hand, if it is a planted plant plant, if drought stress is applied by placing it in a dry environment for about 1 to 2 weeks, and low temperature stress is also applied by placing it in the low temperature environment for about 1 to 2 weeks. Good. In general, since the expression level of a gene introduced into a chromosome varies depending on the position of introduction, the transformed plant individual and its progeny in which the DNA of the present invention is more strongly expressed are selected by performing the analysis described above. it can.

  In addition, the expression of the DNA of the present invention in the transformed plant individual of the present invention or a progeny individual thereof can be finally confirmed and evaluated by examining the oxidative stress resistance of the individual. Such investigation is performed by, for example, applying oxidative stress by cultivating the transformed plant individual of the present invention or a progeny individual thereof together with a non-transformed individual in a culture soil containing a high concentration of salts for 2 to 4 weeks. It can be done by observing the growth situation at the time.

  The protein of the present invention contains at least the DNA of the present invention, that is, the DNA encoding the protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. DNA encoding a protein having an amino acid sequence in which one amino acid has been deleted, substituted, and / or added, DNA having a base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO: 1 or sequence It is encoded by DNA that hybridizes with the base sequence shown in No. 3 under stringent conditions. Such a protein can be obtained from the culture of the transformed cell of the present invention. Here, the culture means cells, tissues, organs, individuals and / or culture media obtained as a result of culturing the transformed cells of the present invention. Culture of the transformed cell of the present invention can be performed using a known method for culturing a cell as the host.

  For example, when culturing transformed cells obtained using microorganisms such as Escherichia coli and yeast as a host, a natural or synthetic medium containing a carbon source, a nitrogen source and inorganic salts is used, usually under aerobic conditions. The shaking culture or the aeration stirring culture may be performed at 37 ° C. for 6 to 24 hours.

  In this case, for example, as a carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, and nitrogen sources include ammonia, ammonium chloride, Ammonium sulfate, ammonium acetate, ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, chloride Sodium, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used singly or in combination of two or more. When culturing transformed cells obtained using plant cells as a host, vitamins such as thiamine and pyridoxine are used. When transforming cells obtained using animal cells as hosts, sera such as RPMI 1640 are used. In addition to the above components, these may be added as necessary. The pH of the medium is preferably adjusted and maintained at 7.0 to 7.5. The pH of the medium can be adjusted using an inorganic or organic acid and / or an alkaline solution.

  In addition, you may add antibiotics, such as penicillin, tetracycline, ampicillin, kanamycin, to the said culture medium in order to prevent the contamination by various bacteria. However, since Escherichia coli usually does not grow in the presence of such antibiotics, addition of these should be avoided when culturing transformed cells using Escherichia coli as a host. However, when an antibiotic resistance gene is used as the selection marker gene and this selection marker gene is introduced into the chromosome together with the DNA of the present invention, the transformed cells are treated with antibiotic resistance corresponding to the introduced antibiotic resistance gene. Therefore, even the transformed cells using Escherichia coli as a host can be cultured by adding the antibiotic to the medium.

  Furthermore, when a compound-inducible promoter is used as a promoter for controlling the DNA of the present invention, it is preferable to add the compound as an inducer to the medium. For example, when the trp promoter is used as the promoter, indoleacrylic acid (IAA) or the like is used, and when the lac promoter is used, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is added to the medium. When the transformed cell of the present invention is cultured, the expression of the DNA of the present invention is induced and the protein of the present invention is accumulated in the culture.

  The protein of the present invention can be obtained by culturing the transformed cell of the present invention as described above and appropriately performing extraction and purification operations on the obtained culture. In addition, when the protein of the present invention is accumulated mainly in transformed cells after culturing, it is preferable to perform an extraction operation after disrupting the cells, and mainly dissolve in the medium after culturing. In the case of accumulating in such a state, it is preferable to carry out the extraction operation after removing the transformed cells and other unnecessary materials from the medium as it is or by centrifugation. For the purification of the protein after extraction, methods known as protein isolation and purification methods, for example, ammonium sulfate precipitation method, gel chromatography method, ion exchange chromatography method, affinity chromatography method, etc. may be used alone or in appropriate combination. Can be used.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples at all.

[Example 1]
I. After extraction and purification of tobacco mRNA, about 10 g of tobacco Nicotiana tabacum L cv. SR-1 leaves grown in a greenhouse for about 1 month were collected, frozen in liquid nitrogen and ground. From this ground product, a crude RNA fraction was extracted using the acid guanidine phenol chloroform method (Chomcznski et al., Anal. Biochem., Vol. 162, p.156-159). After being dissolved in sterilized water treated with diethyl dicarbonate and heated at a temperature of 65 ° C. for 5 minutes, a double equivalent of a loading buffer (20 mM Tris-HCl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0 .1 W / V% SDS) was applied to an oligo dT-cellulose column that had been activated. For purified mRNA, the column is washed with 5-10 equivalents of the above loading buffer, then 2-3 equivalents of elution buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.05 W). / V% SDS) as an eluent. The amount of purified mRNA obtained at this time was 10 μg.

II. Preparation of tobacco cDNA phage library Double-stranded cDNA was synthesized from the purified mRNA obtained in I using Stratagene's HybriZAP 2.1 XR Vector Cloning Kit, and the resulting double-stranded cDNA was used as a λ phage expression vector. After ligation, this expression vector was packaged into phage particles to produce a tobacco cDNA phage library with a titer (titer) of 1.0 × 10 ⁶ pfu / ml.

The phage particles were packaged using an in vitro packaging kit (GigapackII Gold packaging extract) manufactured by Stratagene. That is, after adding Freeze / Thaw extract immediately after lysis to λ phage expression vector linked with tobacco cDNA and placing on ice, immediately add 15 μl of Sonic extract, mix well by pipetting, and then lightly centrifuge, This was carried out by leaving it at room temperature (22 ° C.) for 2 hours. The titer was measured by adding 500 μl of phage dilution buffer (Phage Dilution Buffer) and 20 μl of chloroform to the reaction solution after being left for 2 hours, mixing, and then adding 2 μl of the aqueous layer to 18 μl of SM buffer. (In 1 liter, NaCl 5.8 g, MgSO ₄ .7H ₂ O ₂ g, 1 M Tris-HCl (pH 7.5) 50 ml, 2 W / V% gelatin 5 ml) is diluted with 1 μl of this diluted solution and 1 μl of the phage stock solution. Each was mixed with 200 μl of a culture solution of E. coli PLK-F ′ of ₆₀₀ = 0.5, incubated at 37 ° C. for 15 minutes, added to 2-3 ml of top agar (48 ° C.), and immediately warmed to about 37 ° C. NZY agar plate diameter 150mm (0.5W / V% NaCl, 0.2W / V% MgSO 4 · 7H 2 O, 0.5W / V% yeast extract, 1.0W / V% NZ-amine , 1.5 / V% agar) was layered, 37 ° C., after overnight incubation, were performed by counting the number of occurrences plaques.

III. About 50,000 phage particles (hereinafter also simply referred to as recombinant phage particles) obtained by amplification of a tobacco cDNA phage library and packaged with a λ phage expression vector ligated with tobacco cDNA were OD ₆₀₀ = The mixture was mixed with 600 μl of a culture solution of 0.5 E. coli PLK-F ′ strain and cultured at 37 ° C. for 15 minutes. Immediately overlaid on a 150 mm NZY agar plate and incubated at 37 ° C. for 5-8 hours. Next, 10 ml of SM buffer was added to each plate and cultured overnight at 4 ° C. with gentle shaking to amplify a tobacco cDNA phage library. The titer measured for this amplified library in the same manner as in II above was 5.0 × 10 ⁹ pfu / ml.

  The amplified library is collected in a polypropylene tube, mixed with chloroform corresponding to 5 V / V% of the total amount, left at room temperature for 15 minutes, centrifuged at 4000 g for 5 minutes, and the supernatant is recovered. Chloroform corresponding to 0.3% of the total amount was added to the supernatant and stored at 4 ° C.

IV. Conversion of tobacco cDNA phage library into plasmid Conversion of tobacco cDNA phage library into plasmid includes 200 μl of tobacco cDNA phage library amplified in III (including 1 × 10 ⁵ or more recombinant phage particles), 200 μl of E. coli XL1-Blue culture solution with OD ₆₀₀ = 0.1 and 1 μl of helper phage R408 having a titer of 1 × 10 ⁶ pfu / ml or more were mixed and cultured at 37 ° C. for 15 minutes, and then diluted 2 times with YT medium. Add 5 ml (0.8 W / V% polypeptone, 0.5 W / V% yeast extract, 0.25 W / V% NaCl, 1.5 W / V% agar), and further shake culture at 37 ° C. for 3 hours. It went by. In addition, the tobacco cDNA incorporated in the plasmid thus obtained was heat-treated at 70 ° C. for 20 minutes, and then centrifuged at 4000 g for 5 minutes to recover the supernatant. And then purified by centrifuging for 5 minutes, and then mixed with 20 μl of the 100-fold diluted solution and 200 μl of E. coli XL1-Blue culture solution with an OD ₆₀₀ = 0.1, and cultured at 37 ° C. for 15 minutes. Infected with.

  After the above culture, 1 to 100 μl of the culture solution is separated and LB plate containing ampicillin (1.0 W / V% polypeptone, 0.5 W / V% yeast extract, 1.0 W / V% NaCl, 1.5 W / V% agar) ) At 37 ° C. overnight, colonies of E. coli that appeared were randomly selected, added with glycerol, and stored at −80 ° C. The plasmid incorporating the tobacco cDNA used in the following operations was isolated from the thus-stored E. coli using the QIAfilter Plasmid Kit Maxi manufactured by Qiagen.

  That is, E. coli stored as described above was grown by culturing overnight at 37 ° C. in 5 ml of 2-fold diluted LB medium, and then cultivating overnight at 37 ° C. in 100 ml of LB medium. After that, the cells were collected by centrifugation at 4000 g for 15 minutes at 4 ° C., and 1 ml of TE buffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) was added to the collected cells, followed by vortexing. From this time, the cells were collected again by centrifugation at 5000 g for 5 minutes at 4 ° C. Then, 10 ml of P1 solution (cell suspension solution) was added to the cells, well suspended, and stirred for 2 minutes with a mixer. Further, 10 ml of P2 solution (cell lysis solution) was added, Stir until clear to dissolve cells, add 10 ml of P3 solution (neutralizing solution) to this solution, shake by hand and stir, then centrifuge at 15000 g for 10 minutes to collect the supernatant did. This supernatant was applied to a column QIAGEN-tip 500 previously activated with 10 ml of QBT solution (column activation solution), washed twice with 30 ml of QC solution (column washing solution), then 15 ml of By flowing a QC solution (column elution solution) through the column, the plasmid incorporating tobacco cDNA is eluted, and 10.5 ml of isopropanol is added to the resulting eluate to precipitate it, followed by centrifugation at 15000 g for 30 minutes. And collected by precipitation. Add 1 ml of 70% ethanol to this precipitate, centrifuge at 15000 g for 3 minutes, recover again, completely remove ethanol, vacuum dry in a desiccator, dissolve in 20 μl of sterilized water and bring to −20 ° C. And saved.

V. Reactive oxygen stress resistance gene screening IV Incorporated tobacco cDNA into a yeast strain DHA 12-2c (MATa ho yap1 :: HIS3 yap2 :: URA3 leu2 trp1 ade2 can1) in which both YAP1 gene and YAP2 gene were disrupted -100) using a lithium acetate method, and 1.2 × 10 ^{6 of} these transformed yeasts were added to a YPD medium (1 W / V% yeast extract, 2W / V% polypeptone, 2W / V% dextrose, 2W / V% agar) were cultured at 28 ° C. for 4 to 7 days to screen for reactive oxygen stress resistance genes.

  As a result, it was possible to obtain two transformed yeast strains that were considered to have grown on the above medium and acquired resistance to active oxygen stress.

VI. Determination of the base sequence of the active oxygen stress resistance gene In the two transformed yeasts derived from the two transformed yeast strains that showed resistance to active oxygen stress in V, the introduced plasmids were extracted, and the base sequence was determined. By analysis, the nucleotide sequences of two types of tobacco cDNAs that imparted reactive oxygen stress resistance to these yeasts were determined and named as CDR1 gene and CDR2 gene. The nucleotide sequence of CDR1 gene is shown in SEQ ID NO: 1, and the nucleotide sequence of CDR2 gene is shown in SEQ ID NO: 3.

  In addition, plasmid extraction from yeast was performed by treating yeast cells with cell walls lysed by zymolyce treatment with proteins and SDS and guanidine thiocyanate, followed by treatment with proteolytic enzymes and RNases. It was. Further, the base sequence of cDNA was analyzed by using a sequencing kit manufactured by Beckman Coulter and DNA Auto Sequencer 2000XL according to the attached instructions. At this time, the forward primer (P7) and reverse primer (P8) of the above sequencing kit were used as sequence primers.

  Using the DNA analysis software GENETYX-WIN (manufactured by Software Development Co., Ltd.) and performing a homology search on the CDR1 gene and CDR2 gene based on the GenBank DNA database, the CDR1 gene is highly homologous to the human insulin-degrading enzyme gene. The CDR2 gene had high homology (69% at the amino acid level) with a protein gene of unknown function derived from Arabidopsis thaliana.

VII. Preparation of transformed tobacco into which active oxygen stress tolerance gene was introduced Plasmids introduced into the above two strains of yeast having resistance to active oxygen stress were extracted in the same manner as VI, and Bam HI fragment containing CDR1 gene or CDR2 gene was extracted. After excision and ligation downstream of the 35S promoter of the plasmid pBI121 for binary vector plant cells, this plasmid was transformed into A. coli using the electroporation method. Introduced into Tumefaciens EHA105 strain, Tumefaciens EHA105 (CDR1) or EHA105 (CDR2) was obtained.

  On the other hand, tobacco Nicotiana tabacum L cv. SR-1 grown under aseptic conditions for about 4 weeks after sowing was prepared as a material for producing transformed tobacco, and the medium vein was removed from the leaves and cut into about 5 mm squares. The obtained leaf pieces were cultured in an LB medium containing the antibiotic kanamycin (100 mg / l) for 2 days at 28 ° C. After immersing it in the culture solution of Tumefaciens EHA105 (CDR1) or EHA105 (CDR2) for about 1 to 3 minutes and removing the bacterial solution adhering with a sterilized paper towel, etc., the MS medium (T Murashige and F. Skoog, Physiol. Plant., 15: 473, 1962) were added 1 mg / l naphthalene acetic acid, 0.1 mg / l benzyladenine, 3 W / V sucrose and 0.25 W / V% gellan gum. Place on a callus induction medium so that the back of the leaf is on top, and 25 ° C., all bright (unless otherwise stated, the explants and plant tissues / plants were cultured under these conditions). The culture was performed for 3 days.

  The leaf pieces after the above culture are added with 0.1 mg / l naphthalene acetic acid, 1 mg / l benzyladenine, 500 mg / l carbenicillin, 100 mg / l kanamycin, 3 W / V sucrose and 0.25 W / V% gellan gum to MS medium. After about 4 weeks, the regenerated adventitious buds were cut out, and the adventitious buds were further added to MS medium with benzyladenine 2 mg / l and naphthalene acetic acid 0.1 mg / l. Then, rooting was carried out by transplanting to a rooting medium supplemented with 500 mg / l carbenicillin, 100 mg / l kanamycin and 0.8 W / V% agar to obtain two lines of transformed tobacco. About 4 weeks after transplanting to the rooting medium, rooted individuals are replanted in pots containing culture soil in which vermiculite (Nihon Refractory Industry Co., Ltd.) and peat moss (Izumi Agricultural Materials) are mixed 1: 2. And grown in a greenhouse at 25 ° C. The two lines of transformed tobacco are A. Lines regenerated from leaf pieces after infection with Tumefaciens EHA105 (CDR1) were named NtCDR1, and lines regenerated from leaf pieces after EHA105 (CDR2) infection were named NtCDR2, respectively.

VIII. Analysis of transformed tobacco The following analyzes were performed on two lines of transformed tobacco NtCDR1 and NtCDR2 prepared in VII.

A. PCR analysis Chromosomal DNA was extracted from the leaves of the above two lines of transformed tobacco using the CTAB method (Doyle JJ & Doyle JL, Focus, 12:13, 1988), and the plasmid pBI121 is retained using this DNA as a template. Polymerase chain reaction (PCR) analysis was performed using a set of oligonucleotide primers designed to bind to a predetermined position of the kanamycin resistance gene, and the chromosomal DNA of these transformed tobacco was subjected to kanamycin resistance gene derived from plasmid pBI121. Was confirmed to exist.

  Therefore, these transformed tobaccos include A.I. A structure derived from pBI121 is introduced into the chromosomal DNA through infection with Tumefaciens EHA105 (CDR1) or EHA105 (CDR2). Together with the kanamycin resistance gene, the NtCDR1 strain contains the CDR1 gene, The CDR2 gene is considered to be introduced into the NtCDR2 line.

B. Northern blotting analysis Total RNA was extracted from the stems of the above two lines of transformed tobacco and non-transformed tobacco (both 8 weeks old) by the acid guanidine phenol chloroform method, and 10 μg of this total RNA was used as a buffer solution at 1 × MOPS. Electrophoresis was performed using a buffer solution (20 mM MOPS / pH 7.0, 5 mM sodium acetate, 0.5 mM EDTA) and a 1.2% agarose gel containing 0.66 M formaldehyde as a migration gel at a voltage of 60 V for 2 hours. It was. Here, the non-transformed tobacco refers to wild-type Nicotiana tabacum L cv. SR-1 (the same applies hereinafter).

  The gel after electrophoresis was blotted onto a nylon membrane filter (Hybond-N, manufactured by Amersham Pharmacia Biotech), and a high-grade probe was prepared using a probe prepared according to the protocol of the non-radioisotope DIG-nucleic acid detection system manufactured by Roche Diagnostics. As a result of hybridization, it was revealed that RNA corresponding to the CDR1 gene was highly expressed in transformed tobacco NtCDR1, and RNA corresponding to the CDR2 gene was highly expressed in NtCDR2. On the other hand, expression of these RNAs was not observed in non-transformed tobacco.

C. Investigation of heavy metal salt tolerance and high salt concentration tolerance in the next generation Seeds obtained by self-pollination of the above two lines of transformed tobacco and non-transformed tobacco seeds with 70% ethanol for 1 minute, 10% hypochlorite Sterilized with sodium acid solution for 15 minutes, further washed with sterilized water three times, and then MS solid medium (0.8 W / V% agar) containing 100 μM cadmium chloride (CdCl ₂ ) or 200 mM sodium chloride (NaCl) After culturing for 4 weeks in a cycle of 25 ° C., 16 hours light period and 8 hours dark period, the lengths of stems and roots of germinated and grown seedlings were measured. The results are shown in Tables 1 and 2.

  As is clear from Tables 1 and 2, the next generation of transformed tobacco NtCDR1 and NtCDR2 has a root length of 1.5 times or more in the medium containing 100 μM cadmium chloride compared to non-transformed tobacco. In the medium containing 200 mM sodium chloride, the stem length is 1.9 times or more and the root length is 4.5 times or more in the medium containing 200 mM sodium chloride. It was demonstrated that these transformants have acquired heavy metal salt tolerance and high salt concentration tolerance by the action of the active oxygen stress resistance gene CDR1 or CDR2 introduced into the chromosomal DNA.

[Example 2]
I. Preparation of transformed tobacco cultured cells into which active oxygen stress resistance gene has been introduced 3 days after planting in LS medium (Nagata Y., Meth. Enzymol., 148: 34-39, 1987) at 25 ° C. and 130 rpm Transformation of A. cigarette Nicotiana tabacum L cv. BY-2 cultured cells 4 ml cultured overnight at 30 ° C By adding 100 μl of Tumefaciens EHA105 (CDR1) or EHA105 (CDR2), and statically culturing at 25 ° C. for 48 hours in the dark, these A.P. Tumefaciens was infected, and then centrifuged at 800 rpm for 1 minute to collect the tobacco cultured cells after infection. The collected tobacco cultured cells were added with 5 ml of LS medium, centrifuged well, centrifuged, and the supernatant was discarded. After repeating the washing operation 5 times, LS medium corresponding to 10 times the amount of cells was added and suspended. After turbidity, 1 ml of the aliquot was separated and spread evenly on a solid medium in which carbenicillin 500 mg / l, kanamycin 100 mg / l and 0.8 W / V% agar were added to LS medium. And static culture. After culturing for 2 weeks, the resulting colonies were further cultivated in a medium having the same composition at 25 ° C. in the dark for another 2 weeks, and the cells grown at this time were transformed with transformed tobacco cultured cells NtCDR1-BY2 and NtCDR2-. Selected as BY2. Here, the transformed tobacco cultured cell NtCDR1-BY2 is an A.I. NtCDR2-BY2 is a strain selected by culturing tobacco cultured cells infected with Tumefaciens EHA105 (CDR1). It is a strain selected by culturing tobacco cultured cells infected with Tumefaciens EHA105 (CDR2).

II. Analysis of Transformed Tobacco Cultured Cells The following analyzes were performed on the two transformed tobacco cultured cells NtCDR1-BY2 and NtCDR2-BY2 selected in I.

A. PCR analysis Chromosomal DNA was extracted from the above-mentioned two lines of transformed tobacco cultured cells using the CTAB method, and a set of each set designed to bind to a predetermined position of the CDR1 gene or CDR2 gene using this DNA as a template. PCR analysis was performed using oligonucleotide primers, and the CDR1 gene was present in the chromosomal DNA of the transformed tobacco cultured cell NtCDR1-BY2, and the CDR2 gene was present in the chromosomal DNA of the transformed tobacco cultured cell NtCDR2-BY2. It was confirmed.

B. Investigation of heavy metal salt tolerance The above two lines of transformed tobacco cultured cells were placed on LS solid medium (0.8 W / V% agar) containing 50 μM or 100 μM cadmium chloride, and left to stand for 3 weeks at 25 ° C. in the dark. Thereafter, the fresh weight was measured, and the growth was compared with the fresh weight as 100 when cultured in the same manner without adding cadmium chloride.

  The results are shown in Table 3. In Table 3, MC7-BY2 is linked to the MC7 promoter (Shirasawa-Seo N. et al., Plant Cell Rep., 24: 155, 2005) instead of the 35S promoter as the promoter of the β-glucuronidase gene. Except that a plasmid for plant cells having a structure similar to that of pBI121 was introduced. Tumefaciens EHA105 (MC-7) is used, and YAP1-BY2 is different from the CDR1 gene or the CDR2 gene except that the YAP1 structural gene isolated from yeast is linked downstream of the 35S promoter. Again, a transformation A. strain into which a plant cell plasmid having the same structure as pBI121 was introduced. Using Tumefaciens EHA105 (YAP-1), both of the above-described transformed tobacco cultured cells NtCDR1-BY2 and NtCDR2-BY2 were infected with tobacco cultured cells, and the resulting transformed tobacco It is a cultured cell.

  As apparent from Table 3, the transformed tobacco cultured cells NtCDR1-BY2 and NtCDR2-BY2 both contain cadmium chloride as compared to the transformed tobacco cultured cell MC7-BY2 into which no active oxygen stress resistance gene has been introduced. There is little growth inhibition when cultured in a medium, and as in Example 1, these transformants also acquired heavy metal salt resistance by the action of the active oxygen stress resistance gene CDR1 or CDR2 introduced into the chromosomal DNA. It was proved that. Moreover, NtCDR2-BY2 is a heavy metal salt comparable to the transformed tobacco cultured cell YAP1-BY2 into which the YAP1 gene that plays a central role in the active oxygen removal system is introduced when cultured in a medium containing 50 μM cadmium chloride. Showed tolerance.

Claims (8)

DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. It encodes a protein having an amino acid sequence in which at least one amino acid is deleted, substituted, and / or added to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and is contained in the chromosomal DNA of the host cell. DNA that imparts hydrogen peroxide resistance to the host cell when introduced. DNA having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. A DNA that hybridizes with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions and imparts hydrogen peroxide resistance to the host cell when introduced into the chromosomal DNA of the host cell. A vector for gene transfer comprising the DNA according to claim 1. A transformed cell in which the DNA according to claim 1 is introduced into the chromosomal DNA. A transformed plant individual obtained by growing and differentiating the transformed cell according to claim 6. A protein encoded by the DNA according to claim 1.